WO2002012532A1 - Processus de production de phospholipides - Google Patents

Processus de production de phospholipides Download PDF

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Publication number
WO2002012532A1
WO2002012532A1 PCT/JP2001/006502 JP0106502W WO0212532A1 WO 2002012532 A1 WO2002012532 A1 WO 2002012532A1 JP 0106502 W JP0106502 W JP 0106502W WO 0212532 A1 WO0212532 A1 WO 0212532A1
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WIPO (PCT)
Prior art keywords
phospholipid
reaction
producing
water
phosphatidylserine
Prior art date
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PCT/JP2001/006502
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English (en)
Japanese (ja)
Inventor
Masashi Sakai
Rika Ebina
Hideyuki Yamatoya
Satoshi Kudo
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Kabushiki Kaisha Yakult Honsha
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Priority to DE60139129T priority Critical patent/DE60139129D1/de
Priority to KR1020037001560A priority patent/KR100820657B1/ko
Priority to EP01984491A priority patent/EP1310563B1/fr
Priority to AT01984491T priority patent/ATE435298T1/de
Priority to IL15421801A priority patent/IL154218A0/xx
Priority to BRPI0113132-0A priority patent/BR0113132B1/pt
Application filed by Kabushiki Kaisha Yakult Honsha filed Critical Kabushiki Kaisha Yakult Honsha
Priority to AU2002229150A priority patent/AU2002229150A1/en
Priority to CA002417597A priority patent/CA2417597C/fr
Publication of WO2002012532A1 publication Critical patent/WO2002012532A1/fr
Priority to IL154218A priority patent/IL154218A/en
Priority to US11/065,974 priority patent/US20050170476A1/en
Priority to US11/654,192 priority patent/US7695944B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6481—Phosphoglycerides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen

Definitions

  • the present invention relates to a method for producing a target phospholipid utilizing a phosphatidyl group transfer reaction (phospholipid base exchange reaction).
  • Phospholipids such as phosphatidylserine (PS) and phosphatidylglycerol (PG) have useful physiological actions and unique physical properties, respectively, and are used for pharmaceuticals, food materials, emulsifiers and the like.
  • phosphatidylserine is expected to be used as a drug for the prevention and treatment of senile dementia and memory impairment
  • phosphatidyldaricyl is an emulsifier
  • phosphatidylascorbic acid is expected to be used as an emulsifier
  • a lipid peroxide inhibitor Have been.
  • a method for producing a target phospholipid using a phosphatidyl group transfer reaction has long been known (Yang, SF et al., J. Biol. Chem., 242, p. 477, '67).
  • Kokusei et al. (Aglic. Biol. Chem., 51, p. 2515, '87) reported that a mixture of egg yolk phosphatidylcholine dissolved in isopropyl ether and an L-serine aqueous solution (including calcium chloride) was added. It is stated that a two-phase reaction with phospholipase D yields a reactant containing phosphatidylserine. In a two-phase reaction, it is generally believed that a phosphatidyl group transfer reaction takes place at the interface between the oil phase containing the starting phospholipid and the aqueous phase containing the receptor.
  • Japanese Patent No. 2,924,302 discloses that a phospholipid preparation containing about 85% of phosphatidylcholine obtained by fractionating soybean lecithin is dissolved in ethyl acetate, and further purified by acetic acid.
  • Activating phospholipase D on a mixture containing an aqueous solution containing corbic acid Describes a homogeneous phase reaction method for obtaining a reactant containing phosphatidylascorbic acid.
  • the solvent organic solvent + water
  • the volume is 6 times or more the amount of phospholipid.
  • a reactor had to be used.
  • calcium is added to accelerate the progress of the reaction, but this calcium quickly forms salts with phospholipids.
  • the calcium salts formed in this way fall into the category of chemically synthesized products in Japan and Europe and are difficult to use in foods.
  • Fujita et al.'S patent Japanese Patent Publication No. 7-164426 discloses an organic solvent (diisopropyl ether, isooctane, cyclohexane, benzene, chloroform-isooctane, n-Hexane, dichloromethane rsooctane) and an aqueous phase (including calcium chloride) containing a receptor having a hydroxyl group and phospholipase D in a form encapsulated in reverse micelles. It is described to produce spatidylserine, phosphatidylglycerol and the like.
  • the present invention establishes a production method that enables simple and high-yield production of a target phospholipid without using an organic solvent or calcium when producing a phospholipid using phospholipase D.
  • the purpose is to do.
  • the object described above is to provide a method for producing a phospholipid utilizing a phosphatidyl group transfer reaction, wherein a raw material phospholipid, a receptor having a hydroxyl group, a phospholipase D, and water are treated with an organic compound.
  • a raw material phospholipid, a receptor having a hydroxyl group, a phospholipase D, and water are treated with an organic compound.
  • Homogenization step '' and reacting the obtained homogenized mixture at 15 ° C to 65 ° C And a “reaction step”.
  • the above-mentioned four components of the raw material phospholipid, the receptor having a hydroxyl group, the phospholipase D, and water are mixed well and further subjected to a homogenization treatment.
  • the obtained homogenized mixture is considered to have a lamellar lyotropic liquid crystal structure.
  • the lamellar lyotropic liquid crystal is a phospholipid bilayer liquid crystal formed by adding water to a phospholipid.
  • the bilayer film sometimes also having a multimolecular film structure
  • the aqueous layer are alternately and continuously arranged.
  • the lamellar lyotropic liquid crystal structure can be confirmed, for example, by microscopic observation of the homogenized mixture under crossed Nicols. Incidentally, the “lamellar lyotropic liquid crystal structure without phase separation” described later is observed as a continuous layered structure, and the “lamellar lyotropic liquid crystal structure with phase separation” is observed as a closed annular structure floating in the aqueous phase. You.
  • the lamella-type lyotropic liquid crystal structure when water is added to the raw material phospholipid during homogenization, the interaction between the hydrophilic groups of the phospholipid is weakened and the crystal structure collapses, and a lamella-type liquid crystal is formed. It is caused by By forming a lamella-type lyotropic liquid crystal structure, water can freely move between the layered structures of the phospholipid, and supply of receptors and enzymes, or removal of the polar head liberated from the phospholipid can be performed. It is thought to be performed efficiently and to allow a transfer reaction.
  • the lamella-type lyotropic liquid crystal structure throughout the homogenized mixture.
  • the respective components are mixed but also the homogenization treatment is performed. Need to do. That is, it is preferable to obtain a high reaction product amount by homogenization so that a lamellar lyotropic liquid crystal structure is observed throughout the structure (mainly a state having a lamellar lyotropic liquid crystal structure).
  • the formation of the lamellar lyotropic liquid crystal structure is affected by the water content in the homogenized mixture.
  • the homogenized mixture is a lamellar (neat) liquid crystal having almost no phase separation.
  • the reaction is particularly high when the reaction is carried out in a state where there is no phase separation or in a state where almost no phase separation has occurred (both are referred to as “lamellar lyotropic liquid crystal structure mainly having no phase separation”). It is preferable because metastasis activity is exhibited.
  • the liquid crystal becomes discontinuous vesicles and floats in the solvent, causing phase separation.
  • the efficiency of contact between the water and the phospholipid is lower than in the case where no phospholipid is present, and therefore the transfer activity is also reduced.
  • the water content is too low, the crystal structure of the phospholipid is maintained, and the lamella-type lyotropic liquid crystal structure cannot be formed over the whole, and as a result, the enzymatic reaction field disappears or the fluidity is reduced. Decreases It is thought that the contact efficiency between the substrate and the enzyme deteriorates, and the enzyme cannot work efficiently.
  • phase separation similar to a two-phase reaction as in the conventional method (rather than phase separation in a lamella-type lyotropic liquid crystal structure, rather than the oil phase and aqueous phase). Separation) may be promoted.
  • the addition of the organic solvent causes various disadvantages as described above, it is preferable not to use the organic solvent even during the homogenization treatment.
  • any of a natural product containing a phospholipid, an extract from the natural product, a purified product of the extract, and a synthetic phospholipid may be used.
  • PC phosphatidylcholine
  • PC phosphatidylethanolamine
  • PE phosphatidylethanolamine
  • the use of soybean lecithin, rapeseed lecithin, egg yolk lecithin, or a purified product thereof is preferable from the viewpoint of securing raw materials and costs.
  • the receptor having a hydroxyl group of the present invention may be any receptor that accepts the phosphatidyl group of the raw material phospholipid in the presence of phospholipase D.
  • Examples include serine, glycerol, L-ascorbic acid, glucose, choline, ethanolamine, 1-amino-2-propanol, 1-orthomethyl-dalcoside and the like.
  • Serine, choline, L-ascorbic acid, glucose, glycerol and the like are preferred from the viewpoint of the yield of the target product, and more preferably serine and glycerol are used.
  • the phospholipase D (hereinafter referred to as “PLD”) is not particularly limited as long as it has a phosphatidyl group transfer activity, and its form is free or chemically modified PLD or ion exchange resin. It can also be used as an immobilized enzyme or the like immobilized on a carrier such as silica gel. Among them, the use of free PLD is preferable because the yield of the reaction product is particularly high.
  • PLD specifically, PLD derived from plants such as cabbage and carrot, PLD derived from microorganisms such as actinomycetes, bacteria, yeast, and mold, or Any of animal-derived PLD and the like can be suitably used.Even if prepared by known methods, commercially available products such as cabbage-derived PLD (Sigma, P7758) and peanut-derived PLD (Sigma, P0515 ), PLD derived from Streptomyces chromofuscus ").
  • a raw material phospholipid, a receptor having a hydroxyl group, PLD, and water are mixed, and the mixture is subjected to a homogenization treatment.
  • the homogenization treatment is to finally apply a physical force to the mixture of the above four to uniformly disperse the components, and the methods include physical stirring and ultrasonic treatment. And the like.
  • a homogenization treatment using a bipro mixer, an automatic mortar, a homomixer, a hiscotron, a food processor, or a sonicator alone or in combination can be employed.
  • the mixture may be kneaded using a microspatel or the like.
  • the mixing and homogenization of the four components may be separate or simultaneous in terms of time.For example, homogenization may be performed each time while mixing the four components one by one, or mixing and dissolving 2-4 in advance. After that, it may be finally homogenized.
  • this homogenization treatment if this homogenization treatment is not carried out, the yield of the target phospholipid to be produced will be significantly reduced. This is because the raw material phospholipid is generally in the form of a hard paste at room temperature, and it is difficult to form a lamella-type lyotropic liquid crystal structure entirely by simply mixing other components. This is probably because the contact ratio of each component is reduced and the yield of the target product is reduced.
  • the receptor having a hydroxyl group may be used by dissolving it in water, or may be used as a powder.
  • PLD is used by dissolving in a small amount of water or used as a powder.
  • PA phosphatidic acid
  • the water content during the homogenization treatment is based on the lamellar lyotropic liquid crystal. Since this influences the phase separation of the structure, and eventually the yield of the phospholipid as a reaction product, it is preferable to adjust this to suppress the phase separation.
  • the preferred range of the water content varies depending on the type of the raw material phospholipid. However, if the water content in the lamella lyotropic liquid crystal structure is 10% by weight to 100% by weight with respect to the raw material phospholipid, phase separation is likely to occur. It is possible to suppress the formation of the particles, and particularly preferably 20 to 60% by weight.
  • the water content in the lamellar lyotropic liquid crystal structure refers to the water content in the homogenized mixture after removing the water separated during the homogenization treatment, and the measurement is performed by a known Karl Fischer method. And the like. Further, the separated water may be removed by decantation or centrifugation (for example, at 150 Xg for about 1 minute).
  • it is difficult to generalize the amount of the receptor to be added since the preferable range of the amount varies depending on the type of the receptor to be used. However, it is generally 0.3 to 1 mol of the raw material phospholipid.
  • the molar amount is preferably from 10 to 10 mol, and particularly preferably from 4 to 8 mol, from the viewpoint of the yield of the reaction product and the workability. That is, the addition of a large amount (more than 10 moles) of the receptor increases the labor for recovering the unreacted receptor, while the addition of a small amount results in the recovery of the desired product. The rate drops.
  • the content when serine is used as the receptor having a hydroxyl group, the content should be 5% by weight to 150% by weight, particularly 50% by weight to 100% by weight, based on the raw material phospholipid.
  • the content is preferably 10% by weight to 200% by weight, particularly preferably 20% by weight to 100% by weight.
  • the amount of PLD to be added is not particularly limited, and may be determined in view of the reaction time in the subsequent reaction step, etc., but generally about 500 to 100 000 units per kg of phospholipid. It is.
  • the temperature conditions during the homogenization treatment are not particularly limited, but it is preferable that the temperature be in the range of 15 to 65. If the temperature is lower than 15 ° C, extra energy is required for cooling, and homogenization becomes insufficient. Above 65 ° C, phospholipids become unstable. Further, at the time of the homogenization treatment, edible fats and oils can be added in addition to the components described above, as long as the lamella lyotropic liquid crystal structure is not broken. As with organic solvents, the addition of edible fats and oils can shift the lamellar lyotropic liquid crystal structure to a two-phase system. However, the addition of a small amount will rather increase the flowability of the homogenized mixture and improve the yield of the target phospholipid.
  • Edible oils and fats include, for example, vegetable oils such as safflower oil, soybean oil, corn oil, rapeseed oil, cottonseed oil, castor oil, safflower oil, sesame oil, olive oil, linseed oil, perilla oil, cocoa butter, coconut oil, and butterflies.
  • Oils, fish oils, lard, animal oils such as tallow, medium-chain triacylglycerol (MCT), and the like.One or more of these can be added to and mixed with the raw material phospholipid to impart fluidity. it can. Among them, it is preferable to use MCT, cocoa butter, soybean oil, or the like because the yield of the target product is improved.
  • the amount of the edible oil / fat varies depending on the type of the raw material phospholipid and the type of the edible oil / fat, but is preferably equal to or less than the raw material phospholipid from the viewpoint of the yield. Is preferred.
  • the phosphatidyl group transfer reaction has been carried out by any of a two-phase reaction, a reverse micelle reaction, and a homogeneous phase reaction.
  • a solvent at least 5 times (volume Z weight) relative to the phospholipid.
  • the present invention does not require the use of an organic solvent, which is convenient for the production of substances, such as food, in which the use of the organic solvent is restricted. good. Further, according to the present invention, the reaction proceeds without adding the calcium ion used in the conventional method. Although the reason for this is not clear, in a two-phase reaction or the like, the reaction is performed in a liquid, whereas in the present invention, the reaction is performed in a liquid crystal. It is presumed that the structure takes a structure that is susceptible to a transfer reaction.
  • the homogenized mixture obtained as described above is reacted in the absence of an organic solvent to produce a target phospholipid.
  • the reaction is preferably carried out at 15 ° C to 65 ° C, particularly preferably at 45 ° C to 55 ° C. At a temperature lower than 15 ° C, the progress of the reaction is not accelerated, and the yield of the target phospholipid is reduced. This is because there is a possibility of causing a problem.
  • the reaction time is not particularly limited, and suitable conditions may be selected in accordance with the reaction temperature, the type of components used, their amounts, and the like.
  • the yield of the target phospholipid and the amount of PA by-products produced From the viewpoint, about 1 to 48 hours is preferable.
  • the reaction may be performed in any state such as standing or stirring.
  • the phospholipid according to the method of the present invention can be performed with stirring using a table-top universal mixer (KINMIX MAJOR, KM-230).
  • KINMIX MAJOR table-top universal mixer
  • a suitable amount of water is mixed with the starting phospholipid to form a lamellar liquid crystal state without phase separation, and water can move freely between the phospholipid layered structures. Therefore, since the supply of a receptor or an enzyme having a hydroxyl group or the removal of the polar head liberated from the phospholipid is efficiently performed, a high transfer activity is achieved, and it is inexpensive, simple, and in good yield. It became possible to obtain phospholipids.
  • the phospholipid obtained according to the present invention can be used as a raw material phospholipid in addition to the by-product PA. And phospholipids obtained in many cases may contain impurities in the receptor and the like. The obtained phospholipid is often a mixture containing impurities such as other phospholipids other than the target phospholipid.
  • the transfer-type phospholipid obtained by the transfer reaction be subjected to an appropriate purification treatment step to remove impurities, but as long as there is no problem in administration or a problem that hinders the effect, it is derived from the raw material. Or may be used while containing impurities in the manufacturing process.
  • the purification method is not particularly limited, and may be performed by a suitable combination of fractionation with a solvent, chromatography and the like according to a conventional method.
  • the thus obtained phospholipid of the present invention can also be administered in the form of pharmaceuticals, foods, cosmetics and the like.
  • a pharmaceutical product that claims the physiological effects of phospholipids it can be orally administered as a solid preparation such as capsules, granules, tablets, powders, etc., or a liquid preparation such as a syrup.
  • a parenteral form such as an injection, an external preparation for the skin, a rectal administration and the like, instead of an oral preparation.
  • excipients such as lactose, starch, crystalline cellulose, calcium lactate, magnesium aluminate metasilicate, and silicic anhydride
  • binders such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, carboxymethylcellulose
  • Disintegrators such as calcium carboxymethylcellulose
  • lubricants such as magnesium stearate and talc, and other pharmaceutically acceptable components
  • other pharmaceutically acceptable components may be used as appropriate.
  • the phospholipid obtained by the method of the present invention as it is or appropriately purified may be used to prepare fats and oils, tablet confectionery, fermented milk, bean jam, seasonings It may be added to foods and drinks such as sprinkles and manufactured by a conventional method.
  • the phospholipid obtained by the method of the present invention can be appropriately blended. If the physiological effects of phospholipids are to be promoted, they should be combined in such an amount that the effects can be obtained while avoiding problems such as overdose. For example, when phosphatidylserine is produced as a phospholipid, an amount expected to be in the range of about 50 mg to 100 mg / day may be appropriately blended.
  • the phospholipid of the present invention may be used as an emulsifier, in which case, a drug, It is preferable to add 0.01 to 10% to foods, cosmetics and the like.
  • a phospholipid mixture containing phosphatidylserine is dissolved in alcohols.
  • the phosphatidylserine is insolubilized by adding a metal salt to the solution, and the phosphatidylserine is easily concentrated from the phospholipid mixture containing phosphatidylserine obtained according to the present invention by separating the insoluble portion.
  • the metal salts used include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, and other metal salts, or natural products rich in these, for example, salt, bitter, brine, dolomite, edible pearls
  • a lithium salt, a sodium salt or a potassium salt from the viewpoint of concentration efficiency, and particularly preferable is lithium chloride, sodium chloride or potassium chloride.
  • These metal salts can be used alone or in combination of two or more.
  • the amount of the metal salt to be added is not particularly limited as long as it can precipitate phosphatidylserine.However, the amount is preferably 0.15 to 10 mmol, particularly 0.5 to 5 mmol per lg of the phospholipid. It is preferable in terms of the recovery rate of phosphatidylserine and the high content of phosphatidylserine in the precipitate.
  • any alcohol that can dissolve the phospholipid mixture is preferably used.
  • lower alcohols such as methyl alcohol, ethyl alcohol, butyl alcohol, propyl alcohol, and isopropyl alcohol are preferable.
  • a mixture of these can be used, but it is particularly preferable to use ethyl alcohol because it is easy to use for food and there are few safety problems.
  • the concentration at which the phospholipid mixture is dissolved in alcohols is not particularly limited, but is preferably at least as large as this mixture can be completely dissolved, and is preferably 1 to 50%, especially 1 to 50% by weight of the alcohols. It is preferable to set it to 2 to 20% from the viewpoint of phosphatidylserine concentration efficiency and operability.
  • the concentration of phosphatidylserine from the phospholipid mixture can be performed, for example, as follows. First, it is prepared by a phosphatidyl group transfer reaction method or the like. A phospholipid mixture containing a phospholipid other than sphatidylserine in a component is dissolved in an alcohol such as ethyl alcohol. At this time, the conditions for dissolution such as the dissolution temperature are not particularly limited, and suitable conditions may be selected according to the types of components of the mixture, their amounts, and the like.
  • phospholipids such as phosphatidylserine and PC and PA are extracted into the solvent layer, but in some cases, some insoluble components are generated. For this reason, metal salts are added after removing insoluble components (precipitates, aggregates, etc.) from the solvent by means such as centrifugation and filtration.
  • the extraction treatment with the alcohol may be repeated several times.
  • the metal salt is added as a powder or after being dissolved in a solvent such as water or alcohol.
  • a solvent such as water or alcohol.
  • suitable conditions may be selected according to the types of components of the mixture, their amounts, and the like.
  • PS may be insolubilized by maintaining the temperature at 10 ° C. to 30 ° C. for 30 minutes or more.
  • the phosphatidylserine insolubilized by the addition of the metal salt can be recovered by means such as centrifugation, filtration, and stationary separation. Further, it can be further purified by known purification means, for example, means such as column chromatography. Since the phosphatidylserine concentrate of the present invention has a remarkably reduced content of other phospholipids and the like, such a purification method can be performed relatively easily.
  • FIG. 1 is a diagram showing the results of examining the optimal amount of MCT added.
  • the vertical axis A indicates the PS production rate (%), and the horizontal axis B indicates the amount of MCT added to the phospholipid (%).
  • Figure 2 is a graph showing the results of examining the optimal amount of MCT added.
  • the ratio of MCT addition to lipid (%), and the horizontal axis D is PS generation rate (%).
  • Fig. 3 is a diagram showing the results of verifying the molar ratio of serine to phospholipid in the homogenized mixture.
  • the vertical axis E is the PS generation rate (%), and the horizontal axis F is the molar ratio of serine to phospholipid. is there.
  • Fig. 4 is a diagram showing the results of verifying the weight ratio of water to phospholipid in the homogenized mixture.
  • the vertical axis G is the PS generation rate (%)
  • the horizontal axis H is the water phospholipid (% by weight).
  • Figure 5 is a diagram showing the results of verifying the weight ratio of water to phospholipids in the homogenized mixture.
  • the vertical axis I is the PS generation rate (%)
  • the horizontal axis J is the water nophospholipid (% by weight).
  • Fig. 6 is a diagram showing the results of verifying the weight ratio of water to phospholipid in the homogenized mixture.
  • the vertical axis K is the PS generation rate (%)
  • the horizontal axis L is the water Z phospholipid (% by weight).
  • FIG. 7 is a diagram showing the PS generation rate at each reaction temperature.
  • the vertical axis M is the PS generation rate (%)
  • the horizontal axis N is the temperature (° C).
  • Example 1 (basic)
  • the obtained reaction product was analyzed by silica gel thin-layer chromatography.
  • 0.18 M calcium chloride 47.9% (mol%) of the total phospholipid was converted to phosphatidylserine. It had been.
  • 44.5% of the total phospholipids was converted to phosphatidylserine, and it is clear that the phosphatidyl group transfer reaction proceeds without adding calcium in this reaction system. became.
  • the homogenized mixture before the start of the reaction was observed with a microscope under orthogonal Nicols, a “lamellar-type lyotropic liquid crystal structure having no phase separation” was observed.
  • the present invention provides a method for performing a phosphatidyl group transfer reaction without using an organic solvent. That is, it is possible to efficiently produce a phosphatidyl group transfer reaction product by homogenizing and reacting an aqueous solution containing a phospholipase D with a receptor having a hydroxyl group in a phospholipid.
  • fat-soluble substances other than organic solvents are added to phospholipids, for example, safflower oil, soybean oil, corn oil, rapeseed oil, cottonseed oil, sunflower oil, safflower oil, sesame oil Vegetable oils such as olive oil, linseed oil, sesame oil, cocoa butter, coconut oil, or animal oils such as butter oil, fish oil, lard, tallow, etc., or glyceride derivatives synthesized using medium chain fatty acids as the starting material. It is possible to improve operability by adding edible oils such as MCT. Example 2 (whether calcium was added)
  • Example 1 it was found that the phosphatidyl group transfer reaction proceeds without adding calcium, but the presence or absence of calcium was examined again.
  • the molar ratio of serine to phospholipid is 1 or 5, and each component is mixed under the conditions of calcium addition and non-addition as shown in Table 1, and kneaded using a microspatula (homogenization treatment). The reaction was carried out at 55 ° C for 18 hours.
  • the molar ratio 1 used a 2.5 M serine aqueous solution
  • the molar ratio 5 used a 4.5 M serine aqueous solution.
  • a PLD—Y1 aqueous solution was added to start the reaction.
  • the amount of the reaction product was measured in the same manner as in Example 1. As shown in Table 2, In the case of addition, the Ps generation rate was slightly smaller than that in the case of addition, but almost the same value was obtained. When the homogenized mixture before the start of the reaction was observed with a microscope under crossed Nicols, a “lamellar-type lyotropic liquid crystal structure with no phase separation” was observed in each case.
  • PA indicates phosphatidic acid
  • PE indicates phosphatidylethanolamine
  • PI indicates phosphatidylinosyl.
  • a mixture obtained by kneading MCT (Panasate 810; glycerin fatty acid ester, manufactured by Nippon Oil & Fats ⁇ ⁇ ) in a weight ratio of 1 to 9% to NATHIN 250 acetone precipitate is used as a raw material phospholipid.
  • 20 mg of serine and 200 [iL of water (0.4 in weight ratio of phospholipids and water) are further kneaded to each mg, and an aqueous PLD-Y1 solution (4.1 units / 15 L) is added.
  • the mixture was kneaded using a microspatula (homogenization treatment) and reacted at 55 ° C for 17 hours. After the reaction was completed, phosphatidylserine was measured by silica gel thin-layer chromatography.
  • Figure 1 is a diagram showing the results of studying the optimal amount of MCT. As shown in Fig. 1, the addition of 1 ⁇ ⁇ to 5 011 ⁇ (9% with respect to the phospholipid) increased the PS generation rate from 34.7% to 40.4% compared to the case without the addition. And the effect of adding MCT was observed. The same reaction was carried out with the mixing ratio of MCT being 10% to 40%.
  • Figure 2 is a graph showing the results of examining the optimal amount of MCT. 1 as shown in Figure 2 In all cases where 0% to 40% MCT was added, the PS production rate improved. From FIG. 1 and FIG. 2, it was considered that the added amount of MCT is preferably 9% to 40%.
  • the PS content in the reaction product was 20% or more, which proved to be a suitable condition for PS production.
  • the PS content is 45% or more, which is considered to be a particularly suitable substrate composition.
  • the molar ratio was 10 or more, even if the amount of serine was increased, the amount of PS produced was not improved, and it was considered that the molar ratio of up to 10 was the upper limit for efficient production of phosphatidylserine.
  • Soybean lecithin NATHIN 250 acetone precipitate or PC80: manufactured by Krokran
  • egg yolk lecithin PL-100 LE: manufactured by Cupie
  • L-serine powder in 50 Omg 200 mg of powder was added, mixed well in a mortar heated to about 6 ⁇ , distilled water was kneaded with the water content shown in Figs. 4 to 6, and kept warm at 55 ⁇ .
  • PLD-Y1 manufactured by Yakult Honsha Co., Ltd.
  • the phosphatidyl group transfer reaction was completed at 55 ° C for 17 hours.
  • the water content in the homogenized mixture was measured by the Karl Fischer method.
  • the obtained reaction product was analyzed by silica gel thin-layer chromatography.
  • the horizontal axis was water soybean lecithin weight% (log scale), and the vertical axis was PS content (%) of the reaction product.
  • Figures 4 to 6 are diagrams showing the results.
  • Table 5 shows the transfer reaction product formation rate after the transfer reaction at each receptor.
  • glycerol, L-ascorbic acid, and glucose were used as receptors, a phosphatidyl group transfer reaction product was generated for each of them (phosphatidylglycol).
  • inositol, ascorbic acid phosphate and trehalose were used, no phosphatidyl group transfer product was produced.
  • PL acetone precipitate
  • the formation of the phosphatidyl transfer product was analyzed by thin-layer chromatography on silica gel.
  • FIG. 7 is a graph showing the PS generation rate at each reaction temperature. As shown in Fig. 7, when NACTIN 250 acetone precipitate with MCT was used as the substrate, the PS generation rate at 45 ° C was 43%, which did not change even when the temperature was further increased. .
  • Example 12 Phosphatidylserine concentration 3
  • Example 10 A 25% saline solution was added to the mixture of the extract prepared in Example 10 to form insoluble matter of PS, and the PS content in the precipitated phospholipid and the amount of PS recovered in the precipitate were measured. .
  • Table 6 shows the relationship between the amount of salt added, the PS recovery rate in the precipitate, and the PS content in the phospholipid.
  • PS was concentrated in the precipitated fraction under all conditions, but especially when the amount of salt added per gram of phospholipid in the extract mixture was within 10 mM or less, PS content is more than 55%, and PS is efficiently Had been concentrated.
  • the amount of salt added was 0.05 mmol or less, the PS recovery rate in the precipitate was 60% or less, and 40% or more was present in the supernatant. From the above results, it is possible to concentrate PS into the precipitate at any amount, but the amount of salt added to 0.1 g of phospholipid dissolved in alcohol is preferably in the range of 0.15 to 10 mmol. It was considered that the addition amount was suitable for practical use.

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Abstract

La présente invention concerne un processus de production de phospholipides par transphosphatidylation, qui consiste à mélanger un phospholipide de départ avec un récepteur possédant un ou plusieurs groupes hydroxyle, une phospholipase D et de l'eau en l'absence de solvant organique, à homogénéiser le mélange résultant et à soumettre le mélange homogène obtenu à une réaction sous une température allant de 15?0C à 650¿C. Ce mélange homogène possède une structure de cristal liquide lyotropique laminaire et il peut donner un phospholipide objectif via une transphosphatidylation sans utilisation de solvant organique ni de calcium.
PCT/JP2001/006502 2000-08-09 2001-07-27 Processus de production de phospholipides WO2002012532A1 (fr)

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KR1020037001560A KR100820657B1 (ko) 2000-08-09 2001-07-27 인지질의 제조법
EP01984491A EP1310563B1 (fr) 2000-08-09 2001-07-27 Processus de production de phospholipides
AT01984491T ATE435298T1 (de) 2000-08-09 2001-07-27 Verfahren zur herstellung von phospholipiden
IL15421801A IL154218A0 (en) 2000-08-09 2001-07-27 Process for the production of phospholipids
BRPI0113132-0A BR0113132B1 (pt) 2000-08-09 2001-07-27 mÉtodo para produzir fosfolipÍdeo por transfosfatidilaÇço.
DE60139129T DE60139129D1 (de) 2000-08-09 2001-07-27 Verfahren zur herstellung von phospholipiden
AU2002229150A AU2002229150A1 (en) 2000-08-09 2001-07-27 Process for the production of phospholipids
CA002417597A CA2417597C (fr) 2000-08-09 2001-07-27 Processus de production de phospholipides
IL154218A IL154218A (en) 2000-08-09 2003-01-30 Process for the production of phospholipids
US11/065,974 US20050170476A1 (en) 2000-08-09 2005-02-24 Method for producing phospholipid
US11/654,192 US7695944B2 (en) 2000-08-09 2007-01-17 Method for producing phosholipid

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JP2000241034A JP4298902B2 (ja) 2000-08-09 2000-08-09 リン脂質の製造法

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WO2020133126A1 (fr) * 2018-12-27 2020-07-02 南通励成生物工程有限公司 Procédé de préparation de phosphatidylsérine par enzymolyse assistée par ultrasons
WO2022227248A1 (fr) * 2021-04-27 2022-11-03 内蒙古铂贝曼科技有限公司 Cristal liquide lamellaire phospholipidique actif, son procédé de préparation et son utilisation

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WO2004107875A1 (fr) * 2003-06-06 2004-12-16 Nagase Chemtex Corporation Composition emulsionnable contenant des phospholipides
DE102004002053A1 (de) * 2004-01-15 2005-08-04 Bioghurt Biogarde Gmbh & Co. Kg Verfahren zur Herstellung von Phosphatidylserin und dessen Reinigung durch Extraktion
US20050158835A1 (en) * 2004-01-21 2005-07-21 Su Chen Preparation of highly polyunsaturated fatty acid-containing phosphatidylserine and phosphatidic acid
ITPD20050164A1 (it) 2005-05-30 2006-11-30 Fidia Farmaceutici Processo per la preparazione e l'isolamento di fosfatidi
KR101122388B1 (ko) * 2009-05-23 2012-03-23 주식회사 고센바이오텍 배추 포스포리파아제 d에 의한 난황 인지질로부터 포스파티딜세린의 생합성 방법
KR101055094B1 (ko) * 2009-05-23 2011-08-08 주식회사 고센바이오텍 양배추 포스포리파아제 d에 의한 난황 인지질로부터 포스파티딜세린의 생합성 방법
CN103555783B (zh) * 2013-10-24 2015-08-12 陕西源邦生物技术有限公司 一种制备磷脂酰丝氨酸的方法
CN104327114A (zh) * 2014-11-06 2015-02-04 江南大学 一种磷脂酰丝氨酸的制备方法
CN111534375B (zh) * 2020-05-26 2022-02-08 内蒙古铂贝曼科技有限公司 一种含水磷脂弹性体的制备方法

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WO2020133126A1 (fr) * 2018-12-27 2020-07-02 南通励成生物工程有限公司 Procédé de préparation de phosphatidylsérine par enzymolyse assistée par ultrasons
WO2022227248A1 (fr) * 2021-04-27 2022-11-03 内蒙古铂贝曼科技有限公司 Cristal liquide lamellaire phospholipidique actif, son procédé de préparation et son utilisation

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ATE435298T1 (de) 2009-07-15
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IL154218A0 (en) 2003-07-31
DE60139129D1 (de) 2009-08-13
KR20030033014A (ko) 2003-04-26
US20050170476A1 (en) 2005-08-04
CA2417597C (fr) 2007-06-26
US20030148477A1 (en) 2003-08-07
BR0113132B1 (pt) 2012-03-06
CN1468314A (zh) 2004-01-14
KR100820657B1 (ko) 2008-04-10
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BR0113132A (pt) 2003-07-15
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